Human Cardiac Fibroblasts

Characterization of CFs

Human fetal CFs were cultivated onto 10 cm-diameter dishes for initial expansion. CFs exhibited a flat and spindle-shaped morphology typical of the fibroblastic phenotype (Fig. 1A). CFs strongly expressed fibroblast markers: CD90 in 97.36 ± 0.14% of cells, vimentin in 98.70 ± 0.06%, α-SMA in 97.19 ± 0.15%, DDR2 in 99.62 ± 0.04%, fibronectin in 97.47 ± 0.04%, and pan-cadherin in 96.06 ± 0.15% (n = 3). Conversely, very few cells expressed cardiomyocyte markers cTnT or α-actinin (2.52 ± 1.33% and 1.35 ± 0.40%, respectively; n = 3), ruling out the presence of cardiomyocytes in this population (Fig. 1B).

Additional characteristics of CFs were defined by high-throughput cell profiling for the co-expression of CD90 and various MSC markers: integrin subunit beta 1 (CD29), CD44, 5'-nucleotidase ecto (CD73), endoglin (CD105), vascular cell adhesion molecule 1 (VCAM1, CD106), PDGF receptor alpha (CD140A), STRO-1, and activated leukocyte cell adhesion molecule (CD166). The expressions of a set of MSC-negative markers (i.e., CD34, CD11b, CD19, CD45, and HLA-DR) and c-Kit (CD117) was also assessed. Although CFs shared some characteristics with MSCs, they presented a specific profile in which MSC markers such as CD140A and STRO-1 were not detected (Fig. 1C).

Similarly, the expression of CSC markers (i.e., ATP-binding cassette subfamily G member 2 (CD338), NK2 homeobox 5 (NKX2.5), ISL LIM homeobox 1 (Isl1), and GATA-binding protein 4 (GATA4)) was evaluated. CFs are also positive for most of the CSC markers but not for CD338. CFs also shared characteristics with CSCs, but the lack of expression of critical CSC markers such as CD338 in CFs distinguished the two lineages. Additionally, the expression of several markers of EPDCs (i.e., CD46, WT1 transcription factor (WT1), T-box 18 (TBX18), and myocyte enhancer factor 2C (MEF2C)) was evaluated, and CFs presented an EPDC-like profile. CFs also expressed some endothelial markers such as intercellular adhesion molecule 1 (CD54), but other endothelial markers were expressed over various patterns: platelet and endothelial cell adhesion molecule 1 (CD31) was detected over a broad intensity range, and VE-cadherin (CD144) was not detected (Fig. 1C).

Fig. 1. Micrographs and immunoprofiling of CFs (Iwamiya, Takahiro, et al., 2020).

Cytokine and Chemokine Levels in Human Primary Cardiac Fibroblasts Cultured under Inflammatory Conditions

Human cardiac fibroblasts were subjected to hypoxic and LPS treatment respectively. Levels of cytokines and chemokines were then measured in the culture media. Principal component analysis (PCA) showed a clear separation of the LPS-treated samples from untreated samples and hypoxic samples (Fig. 2a). LPS treatment resulted in an increase in all cytokines/chemokines with the exception of IL-4 and MDC (Fig. 2b). Interestingly, a further separation of the LPS group into two subgroups was seen (Fig. 2a). No clear separation of the hypoxia-treated and untreated samples was observed.

Fig. 2. Principal Component Analysis (PCA) analysis of cytokine and chemokine levels (Sandstedt, Joakim, et al., 2019).

To confirm these findings, two-way ANOVA was carried out for each of the included cytokines/chemokines (Fig. 3). Taken together, the LPS group had a significantly higher production of most of the analyzed cytokines/chemokines compared to control samples and hypoxia-treated samples. Only production of IL-4 was significantly lower in the LPS group compared to the two other groups. No significant differences were observed between the control samples (no LPS, normoxic conditions) and hypoxia-treated samples.

Fig. 3. Graphs showing the mean expression levels for different conditions (Sandstedt, Joakim, et al., 2019).